Experimental animals are indispensable in studies to deeply understand healthy or pathological conditions and to evaluate the efficacy of a candidate treatment targeting application to human diseases. Mice are the experimental animals used most commonly in life science studies. Recently, there are a lot of genetically engineered mouse strains that have been produced to decipher the function of genes as life phenomena and disease models.
A great number of genetically engineered mice are preserved in mouse resource banks, and they are available using the website of International Mouse Strain Resource (IMSR). In mouse resource banks, various reproductive techniques are used, and genetically engineered mice are preserved, transported or produced in an efficient way. To date, an efficient mouse resource bank system is constructed by cryopreservation of sperm, oocytes/ova (hereinafter collectively called “ova”) and embryos of a genetically engineered mouse. Sperm, ova and embryos cryopreserved in mouse resource banks can be easily transported to researchers in need of a genetically engineered mouse by using courier services.
In addition, the present inventors developed a novel technique for conveniently transporting non-frozen mouse embryos or sperm at 4° C. Owing to this technique, special techniques and insulating containers (dryshippers) handling cryopreserved samples became unnecessary. Further, the present inventors developed an in vitro fertilization system using methyl-β-cyclodextrin (MBCD) and reduced glutathione (GSH) (Non-Patent document 1, Non-Patent document 2, Patent document 1, Patent document 2). This technique supports use in medicinal and pharmaceutical fields. Such an in vitro fertilization method can stably attain high fertilization rate using cryopreserved or refrigerated mouse sperm. An improvement in the reproductive techniques is extremely important for carrying out a study efficiently using a genetically engineered mouse.
The phenomenon of superovulation includes follicular maturation and induction of ovulation due to hormonal administration. Superovulation is generally utilized for producing a genetically engineered mouse using various reproductive techniques, and is routinely used for obtaining ova from oocyte/ovum (hereinafter collectively called “ovum”) donors prior to in vitro fertilization (IVF). In superovulation of a mouse, administration of equine chorionic gonadotropin (eCG) promotes growth of a follicle and administration of human chorionic gonadotropin (hCG) induces ovulation. In general, equine chorionic gonadotropin (eCG) stimulating growth of a follicle is administered intraperitoneally to a female mouse, then, human chorionic gonadotropin (hCG) inducing ovulation is injected intraperitoneally (for example, Patent document 3). Equine chorionic gonadotropin (eCG) is one of gonadotropic hormones and called also PMSG, and manifests an action like follicle stimulating hormone (FSH) which is likewise one of gonadotropic hormones. The number of ovulated ova in a C57BL/6 strain which is usually used as the background of a genetically engineered mouse is usually about 9 per female mouse, while it becomes about 25 by a superovulation treatment using eCG and hCG (Non-Patent document 3, Non-Patent document 4).
Administration of inhibin antiserum (IAS) is reported to increase the number of ovulated ova in various animals such as, for example, hamsters, rats, guinea pigs, cows, mares and the like similar to other superovulation treatments (Non-Patent document 5, Non-Patent document 6, Non-Patent document 7, Non-Patent document 8, Non-Patent document 9, Patent document 4). Inhibin is known as a hormone which is secreted from a granulosa cell in the ovarian follicle. Inhibin secreted into the general circulation and acts on the anterior pituitary gland, thereby inhibiting secretion of follicle stimulating hormone (FSH) from the gland. Administration of inhibin antiserum to female rats neutralizes the function of inhibin, resulting in negating negative feedback to FSH by inhibin, and promotes growth of a follicle and the number of ovulated ova in the rat.
It is reported that inhibin antiserum increased the number of ovulated ova in a ddY strain female mouse (Non-Patent document 10, Non-Patent document 11). The effect of inhibin antiserum (IAS) is reported also in a wild-derived strain female mouse (Non-Patent document 12). Immunity neutralization of endogenous inhibin by IAS is an effective strategy for inducing superovulation in a mouse by suppressing negative feedback, thereby increasing endogenous FSH.
Wang et al. first reported that administration of 200 μL of IAS instead of eCG increased the number of ovulated ova in ddY strain immature mice and adult mice. Medan et al. reported that the concentration of FSH in plasma increased remarkably in a ddY strain female mouse treated with IAS. An increase in FSH contributes to promote growth of a follicle and to increase the number of ovulated ova.
Hasegawa et al. likewise report that 50 μL of IAS increases the number of ova in Japanese wild-derived strains (MSM/Ms and JF1/Ms) belonging to Mus musculus molossinus showing low responsiveness to eCG. It is known that for mice of most strains, about 20 to 40 ova can be obtained from one female mouse by promoting maturation of a lot of follicles by eCG having FSH-like action, then, inducing ovulation by hCG, while in many wild-derived mice, this system does not work well. Then, Hasegawa et al. succeeded in increasing the ovulation number of about 5 up to an average of 25 in wild-derived mice MSM/Ms and JF1/Ms by administering anti-inhibin antibody, thereby suppressing negative feedback, to increase endogenous FSH, supposing that the wild-derived mice do not respond to a hormone derived from another species, as one cause thereof.
As described above, the method using anti-inhibin antibody tries to increase the number of ovulated ova by increasing endogenous FSH, instead of excessively administering eCG having FSH-like action from outside (exogenous) to promote growth of a follicle. This would be effective particularly against wild-derived mice for which the remarkable effect is not obtained by eCG, as described above.
According to Hasegawa et al., the age in weeks of a female mouse most suitable for IAS administration is 5 to 7 weeks of age for MSM/Ms and JF1/Ms. Recently, Mochida et al. compared effects of eCG or IAS against the number of ovulated ova in various wild-derived strains from 5 subspecies of Mus musculus (Non-Patent document 13). The responsiveness to eCG or IAS was strong depending on the genetic background thereof. According to Mochida et al., a female mouse belonging to Mus musculus molossinus had a tendency of showing higher responsiveness to IAS as compared with eCG. In contrast, Mus musculus domesticus had a tendency of showing lower responsiveness to IAS as compared with eCG.
As described above, the effect of IAS is reported for some mouse strains, but there is no report on the effect of inhibin antiserum against a C57BL/6 strain mouse which is used most widely as an inbred mouse. This may be based on a fact that a C57BL/6 strain mouse shows high responsiveness to administration of eCG which is an exogenous gonadotropic hormone derived from another species. Further, the reason for this is that Mus musculus domesticus is believed to have lower responsiveness to inhibin antiserum (IAS) as compared with eCG, as described above, according to Mochida et al., and C57BL/6 being known to belong to Mus musculus domesticus. 
The increase of ovulated ova by a superovulation treatment definitely decreases the number of ovum donors and raises the efficiency of animal production. In animal tests, it is important to minimize the number of animals based on the rule of 3 Rs (Reduction, Refinement and Replacement). Thus, it is strongly desired to develop a further efficient novel superovulation technique to increase the number of ovulated ova.